Hello,
I am working with a single exon gene, so there wont be any splice variants. But I would like to design exon-exon spanning primers to check genomic DNA contamination in my RNA. With only one exon in my gene how can I check for genomic DNA contamination. All the above questions are in perspective with RT-qPCR experiment.
You can do either of these: 1) treat your RNA with DNase I prior to cDNA synthesis, this will degrade your genomic DNA in RNA sample. 2) set-up a negative-RT qPCR, in which you add your RNA sample instead to cDNA into the qPCR reaction. If your RNA sample contains gDNA, it would give amplification.
Hi Vishnutej Ellur
since DNA is more robust than RNA even with DNase treatment, yes it's better to use exon spanning (for one of the 2 primers) as one of the rules when designing primers for RTq-PCR, almost you have only one exon in your gene. When designing exons spanning primers, make sure to be at 5' of the gene to avoid RNA degradation bias and that the primers you designed represent well the transcript you targeted. you can also use primer blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome) to see if your primers are really specific.
fred
No RT control is the easiest way to go: you should do this routinely, ideally.
Just a standard cDNA reaction but without reverse transcriptase, which you then treat exactly as you would you your actual cDNA. Run your qPCR using both samples (+RT and -RT), and see what Cq values you get.
It is important to note that you may well get some amplification in your -RT sample, because genomic DNA contamination is pretty hard to avoid, but this is not necessarily a problem. If your +RT Cq values are ~22-24 while your -RT Cq values are 30-32, you can just ignore the issue of gDNA contamination.
My usual benchmark is that +RT Cq values should be at least 6 cycles lower than -RT Cq values: six cycles equates to a 64-fold difference, i.e. your gDNA represents only ~1.5% of your signal. Simple pipetting errors generally contribute significantly more to variability than this, so you can safely ignore this gDNA contribution.
If you find your -RT controls give Cq values comparable to, or within 6 cycles of, your +RT, then maybe DNAse treatment is necessary.
Thank you all. These answers will surely help me. One more question.
My understanding is s single exon gene has no splice variants as there is no need of splicing. Is this assumption correct or am I missing anything ?
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